Ras proteins play an important role in transporting signals from membrane receptors of growth factors to the nucleus, triggering transcription of genes involved in cellular proliferation. They are membrane-bound, guanine nucleotide-binding proteins with GTPase activity. Ras proteins play a role in normal cell function; however, many cancers show an increased expression of normal or mutated Ras proteins that correlate with neoplastic transformation and enhanced proliferation. For Ras proteins to be functional, they must be transported and inserted properly within the cell membrane. Three major post-translational processing events take place which involve the modification of the conserved C-terminus CaaX motif of these proteins: 1) isoprenylation [addition of a farnesyl or geranylgeranyl lipid to the cysteine thiol group], 2) endoproteolytic cleavage of the three carboxyl-terminal amino acids (-aaX), and 3) carboxymethylation of the isoprenylcysteine. Because activated Ras proteins are frequently implicated in many human cancers, these necessary processing steps are attractive potential targets for innovative cancer therapies. Farnesyl transferase inhibitors have limited success because two alternative isoprenylation pathways exist. We are interested in blocking the endoproteolytic processing or methylation steps, as these later steps are shared by farnesylated and geranylgeranylated CaaX proteins alike. The major goal of this study is to test the hypothesis that proteins within the Ras superfamily actually show defective processing in mouse embryonic fibroblasts derived from "knockout" mice lacking a functional CaaX-specific endoprotease or isoprenyl methyltransferase. A second, related goal is to identify and determine if other cellular proteins with CaaX motifs, can serve as alternative protein substrates for the specific endoprotease and isoprenyl methyltransferase. During the next three years we plan to achieve these goals using several proteonomic tools, such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), Western blots probed with specific antibodies and mass spectrometry (MS) protein sequence analysis. The attainment of these goals should pave the way toward the development of CaaX-specific endoprotease and isoprenyl methytransferase inhibitors that may show efficacy in the treatment of Ras-induced cancers.